The complement-mediated lysis of guinea pig erythrocytes by cobra venom factor (CVF) decreased by 50-60% within 2 min of treatment with 5 mM sodium periodate at 0 degree C. This loss of activity paralleled modification of 3-4 Met; other amino acids and sugar residues of the oligosaccharide chains were not affected. Treatment with N-chlorosuccinimide or chloramine-T under conditions that specifically modified 3-4 readily-oxidizable Met also caused 50-60% loss of CVF activity. The secondary structure of CVF was not altered by these modifications. Methionine-modified CVF (MetCVF) supported the cleavage of factor B by factor D with equal efficiency as that of untreated CVF to form C3/C5 convertase (MetCVF,Bb) of the alternative pathway. MetCVF,Bb and CVF,Bb were indistinguishable with respect to C3 cleavage. However, the C5-cleavage ability of MetCVF,Bb was significantly lower than that of CVF,Bb. These results suggest the involvement of Met in CVF binding of C5.